The present invention relates to novel methods and kits for detecting fungal infections, particularly invasive fungal infections, which are based on ergosterol as a biomarker for the presence of fungi. The methods and kits can be utilized for medical diagnosis of a broad spectrum of fungal infections, as well as for detecting fungi in the environment, in food products and in other substances.
The incidence of fungal infections and mycoses has increased significantly in the past two decades, mainly due to the growing number of individuals who have reduced immunological function (immuno-compromised patients), such as cancer patients, patients who have undergone organ transplantation, patients with AIDS, patients undergoing hemodialysis, critically ill patients, patients after major surgery, patients with catheters, patients suffering from severe trauma or burns, patients having debilitative metabolic illnesses such as diabetes mellitus, persons whose blood is exposed to environmental microbes such as individuals having indwelling intravenous tubes, and even in some elderly individuals. Fungal infections are often also attributed to the frequent use of cytotoxic and/or antibacterial drugs, which alter the normal bacterial flora.
Fungi include moulds, yeasts and higher fungi. All fungi are eukaryotic and have sterols but not peptidoglycan in their cell membrane. They are chemoheterotrophs (requiring organic nutrition) and most are aerobic. Many fungi are also saprophytes (living off dead organic matter) in soil and water and acquire their food by absorption. Characteristically fungi also produce sexual and asexual spores. There are over 100,000 species recognized, with 100 infectious members for humans.
Human fungal infections are uncommon in generally healthy persons, being confined to conditions such as Candidiasis (thrush) and dermatophyte skin infections such as athlete's foot. Nevertheless, yeast and other fungi infections are one of the human ailments which still present a formidable challenge to modern medicine. In an immuno-compromised host, a variety of normally mild or nonpathogenic fungi can cause potentially fatal infections. Furthermore, the relative ease with which human can now travel around the world provides the means for unusual fungal infections to be imported from place to place. Therefore, wild and resistant strains of fungi are considered to be one of the most threatening and frequent cause of death mainly in hospitalized persons and immuno-compromised patients.
Invasive fungal infection (IFI) is a serious and potentially life threatening disease that affects a growing number of patients. The projected average incidence of systemic fungal infections in the United States is 306 per million, with Candidiasis accounting for 75% of the reported cases [see, for example, Wilson et al. Value in Health, 5, 26-34, 2002].
Mortality rates in cancer patients who develop systemic fungal infections are very high. It has been observed that fungi are the most common cause of nonbacterial infection in patients with leukemia and lymphoma, with Candida species and Aspergillus being the most common fungal species in cancer patients. These two infections are estimated to have a combined mortality of 20% (Lopez-Berestein et al., Cancer Drug Delivery, 1:37-42, 1983). In other cases, fungal or fungus-like infections, usually introduced into the lungs through the air, are commonplace among large numbers of persons due to environmental exposures.
Certain other organisms that have parasitic properties, such as leishmaniasis, can mimic many of the disease-causing properties, behaviors, and pathologies of fungal infections.
Accurate data regarding the incidence of systemic mycoses and associated mortality are difficult to obtain because reporting requirements vary; many fungal-related deaths are not reported as such because they are undiagnosed, misdiagnosed, or not specified as cause of death. Nevertheless, many indications suggest that the incidence of fungal infections and their attributable mortality are rising. This reflects the increasing number of susceptible hosts due to factors such as the HIV epidemic, advances in organ transplantation and cancer chemotherapy, and the increasing use of invasive procedures for treatment, monitoring, and life support. Estimates are that among the 35 million patients admitted to hospitals in the United States each year, at least 2.5 million will develop nosocomial infections. Almost 250,000 of these will be bloodstream infections, which contribute significantly to excess length and cost of hospital stays and patient mortality. The attributable mortality from bloodstream infections averages 26% but varies according to the specific organism involved.
Of all the pathogens isolated, Candida had the highest attributable mortality rate (40%) [Edmond et al. Clin. Infect. Dis. 29, 239-244, 1999]. Data collected by the NNIS (National Nosocomial Infections Surveillance System) showed that between 1980 and 1989 the incidence of nosocomial candidemia increased by almost 500% in large teaching hospitals and by 219% and 370% in small teaching hospitals and large non-teaching hospitals, respectively [Banerjee et al. Am. J. Med. 91 (suppl. 3B), 86S-89S, 1991]. For an overview of invasive Candidiasis see, for example, http://www.doctorfungus.org/mycoses/human/candida/InvasiveOverview.htm.
Invasive fungal infections therefore pose a major challenge for the management of immuno-compromised and other patients. Currently, mortality rates are high and effective treatment is hampered by the lack of reliable early diagnosis. Since the clinical symptoms of IFI are non-specific, with fever often being the only symptom at the outset, there is a widely recognized need for diagnosis methods that would allow early diagnosis of IFI and thereby would improve the medical outcome and survival of these patients.
Current diagnoses of fungal infections include conventional microbiological, histological and radiological techniques. These techniques, however, are often insufficiently sensitive and have a limited impact on clinical decision-making [Pasqualotto and Denning Europ. Oncology Rev. 1-11, 2005].
The current “gold standard” diagnostic method for fungal infection remains culturing of affected tissue or blood. These cultures are inadequate as they very often fail to grow. It is commonly accepted that blood cultures are positive in less than 50% of patients with autopsy-proven systemic fungal infection [Rodriguez et al. Adv. Pharmacol. 37, 349-400 (1997)]. A recent large retrospective study even suggests that 75% of IFI cases were not found antemortem [Chamilos et al. Haematologica, 91, 986-989 (2006)].
In an attempt to answer the needs for the diagnosis of this elusive group of diseases, various studies have focused on developing new tests. These include, for example, serologic tests and direct blood tests.
Serologic tests (i.e., the detection of specific antibodies to the disease) are difficult to interpret, a feature that often leads to false positive or negative diagnoses. In many cases, since the hosts are immuno-compromised to begin with, serological tests yield no results whatsoever.
Direct blood tests are currently expected to challenge the culturing method and eventually become the gold standard. While still limited in many ways, these tests detect specific antigens, DNA segments, or enzymes present in the blood during the early stages of IFI. Although promising, these tests are presently limited by insufficient specificity, are difficult to deploy since they require expensive infrastructure or laborious preparations, are sometimes difficult to interpret, have cross-reactivity in various clinical settings with common therapies, and are in many cases, prohibitively expensive.
A characteristic commonly shared by organisms that cause all of the above diseases is the presence of ergosterol as the predominant or sole sterol in place of cholesterol.

Ergosterol, a steroid and a precursor to Vitamin D2, is a major component of fungal cell membranes, serving the same function that cholesterol serves in animal cells. Ergosterol is either absent or a minor component of higher plants. The presence of ergosterol in fungal cell membranes coupled with its absence in animal cell membranes makes it a uniquely useful target for fungal diagnostics and antifungal drugs.
In a study of the relationship between viable mould count, ergosterol content and ochratoxin A formation, it was shown that ergosterol assay is useful in the detection of fungus [Saxena et al., Int. J. Food Microbio 71, 29-34 (2001)]. Measurement of ergosterol content was developed as a new method for susceptibility testing of drugs [Arthington-Skaggs et al., Antimicrob. Agents Chemother. 44, 2081-5 (2000)]. Ergosterol determination has also recently been used for determining airborne fungi [Robine et al. J. Microbiol. Meth. 63, 185-192 (2005)], and for determining fungi in environmental samples [Volker, et al. J. Chem. Ed. 77, 1621-3 (2000)].
Hitherto, ergosterol has never been used as a biomarker for systemic fungal infection, probably because appropriate isolation and analytical methods for determining ergosterol levels in clinical samples are not available up to now [Parsi and Gorecki J. Chromatogr. A 1130(1), 145-50 (2006)]. Current methods for ergosterol detection are based on HPLC, mass spectrometry and other analytical instrumentation, which are usually not available in clinical laboratories, are time-consuming and require skilled personnel.
While clinical laboratories often use immunoassay methodologies for diagnosis, these methodologies are ineffective for detecting ergosterol. Ergosterol, as other sterols, is a small, lypophilic molecule, which is typically present in inner membranes. It is well-known in the art that producing effective, specific and sensitive antibodies for such substances, which could be utilized in in vitro diagnoses, is highly difficult and often impossible. Ergosterol is therefore considered as a non-immunogenic molecule [Tejada-Simon and Pestka J. Food Protection 61, 1060-3 (1998)].
One way to overcome the non-immunogenicity of sterols is by conjugation to carrier molecules. However, it is well-known that antibodies generated against sterol compounds conjugated to carrier molecules often cross-react to varying degrees with sterols having similar structures. The basis for cross-reactivity of such antibodies lies in the fact that all of the target compounds against which the antibodies are directed have a similar cyclopentanoperhydrophenanthrine-like multiple ring sterol structure.
U.S. Patent Application Publication No. 20020018808 teaches liposomal or other delivery compositions that contain ergosterol or ergosterol derivatives, and methods of using same. These compositions are useful for immunizing humans and animals against fungal infections and for the treatment and prevention of fungal infection. U.S. Patent Application No. 20020018808 further teaches diagnostic assays and kits for determining whether a human or animal has a fungal infection by measuring antibodies to ergosterol, whereby these assays and kits utilize plates having ergosterol or anti-ergosterol antibodies bound thereto.
While U.S. Patent Application No. 20020018808 suggests a synthetic pathway for preparing N-[(3β,22E)-ergosta-5,7,22-trien-3-(succinylamido)]dimyristoyl-phosphatidyl ethanolamine, a phosphatidyl ergosterol, to be encapsulated within liposome, so as to serve as a vaccine composition, the use of such ergosterol-containing liposomes and the efficient production of antibodies against ergosterol upon administering these liposomes are not described.
1,2,4-Triazoline-3,5-dione (TAD) and derivatives thereof are well-known dienophiles which have been widely utilized and studied in the Diels-Alder reaction with 1,3-diene-containing compounds, including substances in biological systems. For example, a 4-N-pentafluorobenzyl-1,2,4-triazoline-3,5-dione was shown to react with an analog of vitamin D3 and it was suggested that this approach could be used for detecting vitamin D2, vitamin D3 and other drugs or biological substances containing a 1,3-diene moiety [see, for example, Wang, et al., Anal. Biochem. 243, 28-40 (1996)]. Thus, for example, a triazolinedione derivative was used to simultaneously determine vitamin D2 and vitamin D3 in plasma [Higashi et al., Biol. Pharm. Bull. 24, 738-743 (2001)].
By including a 1,3-diene moiety, ergosterol has been used in various studies concerning Diels-Alder reactions of sterols and various dienophiles (see, for example, U.S. Pat. No. 6,399,796). Thus, conjugates of ergosterol and triazolinedione derivatives have been reported. For example, an adduct of 4-phenyl-1,2,4-triazoline-3,5-dione and ergosterol was prepared [Gilani and Triggle, J. Org. Chem. 31, 2397 (1966)]. These conjugates, however, have never been utilized in diagnostic methods for detecting fungal infections by measuring the level of ergosterol.
As discussed hereinabove, despite recent advances in treatment, mortality rates of invasive fungal infections remain unacceptably high, particularly with regard to the two most common pathogen groups, Candida and Aspergillus. Fast, accurate diagnosis remains a key obstacle in the treatment of invasive as well as other fugal infections.
There is thus a widely recognized need for, and it would be highly advantageous to have, an ergosterol-based biomarker for the accurate, fast and specific detection of fungi and methods and kits utilizing same, devoid of the above limitations.